Cancer Letters

Cancer Letters

Volume 383, Issue 2, 28 December 2016, Pages 282-294
Cancer Letters

Original Article
Glutaminase 2 stabilizes Dicer to repress Snail and metastasis in hepatocellular carcinoma cells

https://doi.org/10.1016/j.canlet.2016.10.012Get rights and content

Abstract

Glutaminolysis that catabolizes glutamine to glutamate plays a critical role in cancer progression. Glutaminase 2 (GLS2) has been reported as a tumor suppressor. Recent studies implied that GLS2 may display its multifunction besides classical metabolic feature by different localizations and potential protein binding domains. Here, we showed that GLS2 expression correlates inversely with stage, vascular invasion, tumor size and poor prognosis in human hepatocellular carcinoma (HCC) tissues. We found that GLS2 significantly represses cell migration, invasion and metastasis of HCC through downregulation of Snail in vitro and in vivo. Moreover, our results demonstrated that GLS2 interacts with Dicer and stabilizes Dicer protein to facilitate miR-34a maturation and subsequently represses Snail expression in a glutaminase activity independent manner. Our findings indicate that non-glutaminolysis function of GLS2 inhibits migration and invasion of HCC cells by repressing the epithelial–mesenchymal transition via the Dicer-miR-34a-Snail axis.

Introduction

Hepatocellular carcinoma (HCC) is one of the most common malignancies with the leading causes of cancer death worldwide [1]. HCC has a poor prognosis due to its rapid development and early recurrence [2]. Therefore, there is an urgent need to understand of the exact molecular mechanisms leading of HCC progression and identify novel therapeutic targets.

Epithelial–mesenchymal transition (EMT) is defined as a complex process and is believed to play important roles in embryonic development, regeneration, wound healing, cancer invasion and metastasis [3]. The EMT regulators overexpress in about 50% of HCC and correlates with recurrence and worse prognosis of patients [4], [5]. MicroRNAs (miRNAs) are a class of endogenous noncoding RNAs molecules of 21–23 nucleotides and capable to repress gene expression by posttranscriptional regulation [6]. MiRNAs play crucial roles in diverse physiologic and pathologic processes including cancer progression [7]. MiRNA biogenesis is depending on sequential direction of the processing machineries, including Drosha, AGO, and Dicer [8]. Mutation and dysregulation of the miRNA biogenesis pathway have been widely observed in cancer [9]. Dicer, an RNase III ribonuclease, is often dysregulated in diverse cancer types and exhibits its function by regulating specific miRNAs [10], [11], [12].

Metabolic deregulation has been recognized an important hallmark of cancer [13], [14]. Warbug effect is the most well-known phenomena which is tumor cells typically exhibit increased aerobic glycolysis. In addition to elevated glycolysis, increased glutaminolysis is another characteristic for malignancies [15], [16]. Glutaminase is the critical enzyme for converting glutamine to glutamate in glutaminolysis [17]. There are two major human glutaminase genes, GLS1 and GLS2, which exhibit distinct tissue distributions and different cellular regulation [18]. Glutaminase1 (GLS1) acts as an oncogene, which has been reported to promote cancer progression and upregulation in diverse tumors [19], [20]. Overexpression of GLS2 reduces proliferation, migration and colony formation in vitro [21], [22], [23] and sensitizes the cells to alkylating agents [24]. GLS2 also exhibits its tumor suppressive ability through downregulation of PI3K/AKT signaling and GLS2 expression associated with higher overall survival of HCC patients [25], [26]. However, silencing GLS2 sensitizes radio-resistant cervical cancer cells to ionizing radiation [27] and represses cell growth in MYCN-amplified neuroblastoma cells, lung cancer and HCC cell lines [28], [29]. Therefore, more works are needed to validate and clarify the controversial role of GLS2 in tumor growth. Recent studies implied the possible function of GLS2 through its different localizations and potential protein binding domains [30]. GLS2 may execute other functions besides glutaminolysis. In addition to cell growth, little is known the role and mechanisms of GLS2 in cancer cell motility.

In this study, we demonstrate a novel mechanism of GLS2-mediated suppression of invasion and metastasis in HCC cells. We show that GLS2 interacts with Dicer and stabilizes it to promote miR-34a maturation. Upregulated miR-34a subsequently represses EMT and HCC motility by inhibiting Snail expression. Moreover, we found that GLS2 represses HCC motility through non-glutaminase ability. These findings indicate that non-glutaminolysis function of GLS2 inhibits HCC cells migration and invasion by repressing the EMT via the Dicer-miR-34a-Snail axis.

Section snippets

Cell lines

The HepG2, C3A, Hep3B and 293T cell lines were purchased from the American Type Culture Collection (ATCC), and Huh1 and Huh7 cell lines were purchased from the Japanese Collection of Research Bioresources Cell Bank (JCRB). HA22T/VGH was obtained from the Bioresource Collection and Research Center of the Food Industry Research and Development Institute (Hsinchu, Taiwan). The Mahlavu and HCC36 cells were cultured as described [31], [32]. All cells were routinely authenticated on the basis of

GLS2 expression inversely correlates with poor prognosis of HCC patients

To determine the clinical significance of GLS2 expression in HCC patients, we analyzed samples from two independent cohorts of HCC patients. Cohorts 1 and 2 included 129 and 88 HCC patients, respectively. GLS2 protein level was determined using immunohistochemistry (IHC) staining in cohort 1, and GLS2 mRNA level was determined using quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay in cohort 2. In cohort 1, GLS2 expression was detectable in normal liver part (Fig. 1

Discussion

Recently, GLS2 had been reported to exhibit the opposite phenotypes in regulation of tumor growth [21], [22], [23], [26], [27], [28], [29]. However, whether GLS2 can influence other biological functions and the exhaustive mechanisms are unclear. Previous studies clearly demonstrated that the downregulation of GLS2 is a common event in HCC tissues as well as in different HCC cell lines, and the lower GLS2 expression was attributed to its promoter hypermethylation [26], [36]. In this study, we

Author contributions

TCK, CKC, KTH and PY performed and interpreted all experiments with the help of WJL, MWC, MHC and KTK. CKC, PY and MWC made constructs. YMJ and MH provided annotated HCC samples. TCK and MLK conceived and designed the experiments and wrote the manuscript. MLK supervised the entire study. All authors contributed to study concept, design and data analyses, drafted and reviewed the manuscript revisions, and approved the final draft for submission.

Acknowledgments

We thank National RNAi Core Facility (Academia Sinica, Taiwan) for providing specific shRNAs. The part clinical results shown here are based upon data generated by the Gene Expression Omnibus (GEO): http://www.ncbi.nlm.nih.gov/geo/ and The Cancer Genome Atlas (TCGA): http://cancergenome.nih.gov. This work was supported by grants for the National Science Council, Taiwan (NSC 101-2320-B-002-042 to M-L Kuo), Ministry of Science and Technology, Taiwan (MOST 104-2321-B-002-006, MOST

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